三疣梭子蟹(Portunus trituberculatus)肝胰腺内源酶性质的初步研究
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摘要
内源酶是三疣梭子蟹(Portunus trituberculatus)体内重要的酶,往往会导致梭子蟹死后肌肉组织迅速软化,严重影响了蟹肉的贮藏品质。为了探明该酶的基本特性,本试验从三疣梭子蟹肝胰腺中提取了粗酶,优化了提取方法,并对其部分酶学性质进行初步研究。结果表明:三疣梭子蟹肝胰腺内源酶最佳浸提时间段为4～12 h,酶比活显著高于0～4 h和12～14 h;以酪蛋白为水解底物,内源酶作用最适温度为65℃、最适p H范围为7.0～8.0;丝氨酸蛋白酶特异性抑制剂,包括大豆胰蛋白酶抑制剂(SBTI)和苯甲基磺酰氟(PMSF),对内源酶活力的相对抑制率分别为100%、70.46%±6.27%,显著高于其他抑制剂的相对抑制率,推测丝氨酸蛋白酶为主要内源酶;在硫酸铵分级沉淀中,分别以酪蛋白和Boc-Phe-Ser-Arg-MCA为底物,前者最适盐析浓度为0～70%,后者为30%～70%,酶比活显著高于其他盐浓度;当硫酸铵浓度为40%～60%时,盐析蛋白质含量、粗酶酶活和丝氨酸蛋白酶活均显著高于其他盐浓度。
Endogenous autolytic enzyme is an important enzyme in Portunus trituberculatus. Normally, after the death of swimming crabs, deterioration is very rapid in the muscle, which produces a severe effect on the storage quality of crabs. To investigate few basic properties of this autolytic enzyme, crude samples were extracted from the hepatopancreas of P. trituberculatus and the parameters are optimized. Then, a preliminary study on the enzymatic properties of the endogenous enzyme was conducted. The results showed that the optimum extraction time range of endogenous protease was 4–12 h, and the specific activity was significantly higher than that at other time points, i.e., 0–4 h and 12–14 h. The optimal temperature and p H for hydrolysis of casein were 65℃ and 7.0–8.0, respectively. Compared with other protease inhibitors, endogenous protease activity was significantly inhibited by serine protease inhibitors, including soybean trypsin inhibitor(SBTI) and phenylmethylsulfonyl fluoride(PMSF), suggesting that serine protease was the dominant content of this endogenous enzyme. Ammonium sulfate grading precipitation showed that fractions of 0%–70% and 30%–70% could be used to collect the targeted proteases for casein and Boc-Phe-Ser-Arg-MCA hydrolysis, respectively. Their specific activities were significantly higher than those of the samples precipitated by other ammonium sulfate fractions. Moreover, samples collected by ammonium sulfate fractions of 40%–60% had a higher protein content and total activity of crude and serine proteinase than those of the samples precipitated by other ammonium sulfate fractions.
Endogenous autolytic enzyme is an important enzyme in Portunus trituberculatus. Normally, after the death of swimming crabs, deterioration is very rapid in the muscle, which produces a severe effect on the storage quality of crabs. To investigate few basic properties of this autolytic enzyme, crude samples were extracted from the hepatopancreas of P. trituberculatus and the parameters are optimized. Then, a preliminary study on the enzymatic properties of the endogenous enzyme was conducted. The results showed that the optimum extraction time range of endogenous protease was 4–12 h, and the specific activity was significantly higher than that at other time points, i.e., 0–4 h and 12–14 h. The optimal temperature and p H for hydrolysis of casein were 65℃ and 7.0–8.0, respectively. Compared with other protease inhibitors, endogenous protease activity was significantly inhibited by serine protease inhibitors, including soybean trypsin inhibitor(SBTI) and phenylmethylsulfonyl fluoride(PMSF), suggesting that serine protease was the dominant content of this endogenous enzyme. Ammonium sulfate grading precipitation showed that fractions of 0%–70% and 30%–70% could be used to collect the targeted proteases for casein and Boc-Phe-Ser-Arg-MCA hydrolysis, respectively. Their specific activities were significantly higher than those of the samples precipitated by other ammonium sulfate fractions. Moreover, samples collected by ammonium sulfate fractions of 40%–60% had a higher protein content and total activity of crude and serine proteinase than those of the samples precipitated by other ammonium sulfate fractions.
引文
[1]董志国.中国沿海三疣梭子蟹群体形态、生化与分子遗传多样性研究[D].上海:上海海洋大学,2012.Dong Zhiguo.The study on morphology,biochemistry,molecular phylogeography and genetic dibersity of the swimming crab Portunus trituberculatus along China coast[D].Shanghai:Shanghai Ocean University,2012.
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[12]Misook K,Yoonhwa J.Purification and characterization of a trypsin-like protease from flatfish(Paralichthys olivaceus)intestine[J].Journal of Food Biochemistry,2013,37(2013):732-741.
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[14]吴志强.太平洋磷虾(Euphausiapacifica Hansen)胰蛋白酶样酶酶学性质及基因片段研究[D].青岛:中国海洋大学,2007.Wu Zhiqiang.Enzymatic characteristics and partial gene of trypsin-like enzymes from Euphausiapacifica Hansen[D].Qingdao:Ocean University of China,2007.
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[16]Jiang S T,Moody M W,Chen H C.Purification and characterization of protease from digestive tract of grass shrimp(Penaeus monodon)[J].Journal of Food Science,1991,56:322-326.
[17]启航,董秀萍,高杨,等.海参体壁粗蛋白酶的提取及酶学性质研究[J].食品研究与开发,2007,28(3):4-8.Qi Hang,Dong Xiuping,Gao Yang,et al.Studies on the extraction and enzymology character of the crude protease from the body wall of holothurians[J].Food Research and Development,2007,28(3):4-8.
[18]庄志凯,吉宏武.南美白对虾虾头内源酸性蛋白酶的分离纯化及酶学特性研究[J].食品工业科技,2012,33(18):116-120.Zhuang Zhikai,Ji Hongwu.Purification and characteristics of endogenous acid protease from shrimp head of Litopenaeus vannanmei[J].Science and Techonlogy of Food Industry,2012,33(18):116-120.
[19]杜翠红,曹敏杰.鱼类肌原纤维结合型丝氨酸蛋白酶研究进展[J].食品科学,2013,34(9):336-339.Du Cuihong,Cao Minjie.Progress in research on myofibril-bound serine proteinase from fish[J].Food Science,2013,34(9):336-339.
[20]Pornpimol S,Chodsana S.A trypsin from royal red prawn(Haliporoides sibogae)and its possible application for collagen hydrolysis[J].Separation Science and Technology,2015,50:1073-1082.
[21]Benjakul S,Visessanguan W,Leelapongwattana K.Purification and characterization of heat-stable alkaline proteinase from bigeyesnapper(Priacanthus macracanthus)muscle[J].Comparative Biochemistry and Physiology,B:Comparative Biochemistry,2003,124:107-127.
[22]Eggerer J.Hysteretic behaviour of citrate synthase:site-directed limited proteolysis[J].European Journal of Biochemistry,1984,143:205-212.
[23]Benjakul S,Leelapongwattana K,Visessanguan W.Comparative study on proteolysis of two species of bigeye snapper,Priacanthus macracanthus and Priacanthus tayenus[J].Journal of the Science of Food and Agriculture,2003,83(9):871-879.
[24]Klomklao S,Benjakul S,Visessanguan W.Comparative studies on proteolytic activity of splenic extract from three tuna species commonly used in Thailand[J].Journal of Food Biochemistry,2004,28(5):355-372.
[25]银凤,周爱梅,张祥刚,等.南美白对虾虾头主要自溶酶的分离纯化及鉴定[J].食品工业与发酵,2011,37(3):23-26.Yin Feng,Zhou Aimei,Zhang Xianggang,et al.Purification and identification of major autoenzyme from the head of white shrimp(Penaeus vannamei)[J].Food and fermentation Industries,2011,37(3):23-26.
[26]王秋岑.不同食性鱼类胰蛋白酶的提纯、性质及消化力的比较研究[D].南昌:南昌大学,2013.Wang Qiucen.Comparative study on proteolytic digestion,purification and characterization of trypins from different feeding fish[D].Nanchang:Nanchang University,2013.
[2]Dionysius D A,Hoek K S,Milne J M,et al.Trypsin-like enzyme from sand crab(Portunus pelagicus):purification and characterization[J].Journal of Food Science,1993,58(4):780-784.
[3]Chodsana S,Soottawat B,Wonnop V,et al.Collagenolytic serine protease in fresh water prawn(Macrobrachium rosenbergii):Characteristics and its impact on muscle during ice storage[J].Food Chemistry,2011,124(2011):29-35.
[4]蔡秋凤,林敏,钟婵,等.口虾蛄肌肉中丝氨酸蛋白酶的纯化及性质分析[J].中国食品学报,2013,13(11):185-190.Cai Qiufeng,Lin Min,Zhong Chan,et al.Purification and characterization of serine proteinase from mantis shrimp(Squilla oratoria)[J].Journal of Chinese Institute of Food Science and Technology,2013,13(11):185-190.
[5]Yan L J,Zhan CH L,Cai Q F,et al.Purification,characterization,c DNA cloning and in vitro expression of a serine proteinase from the intestinal tract of sea cucumber(Stichopus japonicus)with collagen degradation activity[J].Journal of Agricultural and Food Chemistry,2011,62(20):4769-4777.
[6]Klomklao S,Kishimura H,Benjakul S,et al.Cationic trypsin:A predominant proteinase in pacific saury(Cololabis saira)pyloric ceca[J].Journal of Food Biochemistry,2010,34(5):1105-1123.
[7]Klomklao S,Benjakul S,Visessanguan W,et al.Purification and characterisation of trypsins from the spleen of skipjack tuna(Katsuwonus pelamis)[J].Journal of Food Chemistry,2007,100(4):1580-1589.
[8]陈毓荃.生物化学实验方法和技术[M].北京:科学出版社,2002:95-97.Chen Yuquan.Biochemical experimental methods and techniques[M].Beijing:The Science Publishing Company,2002:95-97.
[9]Jensen S E,Philipe L,Stemke G W,et al.Purification and characterization of exocellular proteases produced by a clinical isolate and a laboratory strain of Pseudomonas aeruginosa[J].Canadian Journal of Microbiology,1980,26:77-86.
[10]Yoshida A,Bae I,Sonoda H,et al.Characterization of gelatinolytic enzymes in the skeletal muscle of red sea bream Pagrus major[J].Fisheries Science,2009,75(5):1317-1322.
[11]Chodsana S,Soottawat B,Wonnop V,et al.Low molecular weight trypsin from hepatopancreas of freshwater prawn(Macrobrachium rosenbergii).Characteristics and biochemical properties[J].Food Chemistry,2012,134(2012):351-358.
[12]Misook K,Yoonhwa J.Purification and characterization of a trypsin-like protease from flatfish(Paralichthys olivaceus)intestine[J].Journal of Food Biochemistry,2013,37(2013):732-741.
[13]Walsh K A.Trypsinogen and trypsins of various species[J].Methods Enzymology,1970,19:41-63.
[14]吴志强.太平洋磷虾(Euphausiapacifica Hansen)胰蛋白酶样酶酶学性质及基因片段研究[D].青岛:中国海洋大学,2007.Wu Zhiqiang.Enzymatic characteristics and partial gene of trypsin-like enzymes from Euphausiapacifica Hansen[D].Qingdao:Ocean University of China,2007.
[15]杭虞杰,李学英,杨宪时,等.南极磷虾自溶酶性质的初步研究[J].食品科学,2011,32(13):198-200.Hang Yujie,Li Xueying,Yang Xianshi,et al.Properties of autolysis enzymes from antarctilkill[J].Food Science,2011,32(13):198-200.
[16]Jiang S T,Moody M W,Chen H C.Purification and characterization of protease from digestive tract of grass shrimp(Penaeus monodon)[J].Journal of Food Science,1991,56:322-326.
[17]启航,董秀萍,高杨,等.海参体壁粗蛋白酶的提取及酶学性质研究[J].食品研究与开发,2007,28(3):4-8.Qi Hang,Dong Xiuping,Gao Yang,et al.Studies on the extraction and enzymology character of the crude protease from the body wall of holothurians[J].Food Research and Development,2007,28(3):4-8.
[18]庄志凯,吉宏武.南美白对虾虾头内源酸性蛋白酶的分离纯化及酶学特性研究[J].食品工业科技,2012,33(18):116-120.Zhuang Zhikai,Ji Hongwu.Purification and characteristics of endogenous acid protease from shrimp head of Litopenaeus vannanmei[J].Science and Techonlogy of Food Industry,2012,33(18):116-120.
[19]杜翠红,曹敏杰.鱼类肌原纤维结合型丝氨酸蛋白酶研究进展[J].食品科学,2013,34(9):336-339.Du Cuihong,Cao Minjie.Progress in research on myofibril-bound serine proteinase from fish[J].Food Science,2013,34(9):336-339.
[20]Pornpimol S,Chodsana S.A trypsin from royal red prawn(Haliporoides sibogae)and its possible application for collagen hydrolysis[J].Separation Science and Technology,2015,50:1073-1082.
[21]Benjakul S,Visessanguan W,Leelapongwattana K.Purification and characterization of heat-stable alkaline proteinase from bigeyesnapper(Priacanthus macracanthus)muscle[J].Comparative Biochemistry and Physiology,B:Comparative Biochemistry,2003,124:107-127.
[22]Eggerer J.Hysteretic behaviour of citrate synthase:site-directed limited proteolysis[J].European Journal of Biochemistry,1984,143:205-212.
[23]Benjakul S,Leelapongwattana K,Visessanguan W.Comparative study on proteolysis of two species of bigeye snapper,Priacanthus macracanthus and Priacanthus tayenus[J].Journal of the Science of Food and Agriculture,2003,83(9):871-879.
[24]Klomklao S,Benjakul S,Visessanguan W.Comparative studies on proteolytic activity of splenic extract from three tuna species commonly used in Thailand[J].Journal of Food Biochemistry,2004,28(5):355-372.
[25]银凤,周爱梅,张祥刚,等.南美白对虾虾头主要自溶酶的分离纯化及鉴定[J].食品工业与发酵,2011,37(3):23-26.Yin Feng,Zhou Aimei,Zhang Xianggang,et al.Purification and identification of major autoenzyme from the head of white shrimp(Penaeus vannamei)[J].Food and fermentation Industries,2011,37(3):23-26.
[26]王秋岑.不同食性鱼类胰蛋白酶的提纯、性质及消化力的比较研究[D].南昌:南昌大学,2013.Wang Qiucen.Comparative study on proteolytic digestion,purification and characterization of trypins from different feeding fish[D].Nanchang:Nanchang University,2013.